Project Summary The primary goal of this application is to elucidate the neural basis of resting-state functional magnetic resonance imaging (rsfMRI) signal using multi-modal approaches including multi-echo (ME)- rsfMRI, MR-compatible calcium signal recording, optogenetics and multi-laminar electrophysiology in awake rats. Despite the prominent role of rsfMRI in studying brain network function in health and disease, the neural basis of rsfMRI signal remains poorly understood. In particular, cellular and circuit-level mechanisms underlying resting-state functional connectivity (RSFC) are unknown. This critical knowledge gap has hampered the interpretation of rsfMRI signal in light of underlying neuronal activity. Here, we propose an integrated strategy to mechanistically dissect the neural basis of RSFC by combining ME-rsfMRI, cell-type specific calcium-based fiber photometry, optogenetics and electrophysiology. Specifically, ME-rsfMRI can differentiate neural and non-neural components in rsfMRI signal. In Aim 1, we will apply ME-rsfMRI to obtain precise RSFC quantification by eliminating non-neural artifacts. Second, calcium-based fiber photometry is an optical method that directly measures spiking activity from a defined neuron population (e.g. excitatory neurons) based on a genetically encoded calcium indicator (GCaMP). Thus, combining calcium-based fiber photometry with rsfMRI offers simultaneous neuronal and rsfMRI signal measurement with neuron-type specificity. In Aim 2, we will use this technique to dissect the contributions of spiking activity from individual neuron populations to rsfMRI signal. This study will provide critical insight into the cellular mechanism underlying rsfMRI signal. We will also determine how cell-type specific calcium signals link to the local field potential and spiking activity measured by electrophysiology. In Aim 3, we will integrate optogenetics, rsfMRI and multi-laminar electrophysiology to determine the causal relationship between RSFC modulation and layer-specific neural activity change. We will modulate RSFC by optogenetically increasing neuronal excitability using Stabilized Step-Function Opsin (SSFO), and examine the resulting RSFC and layer-specific electrophysiology signal changes. Linking multi-laminar electrophysiology and rsfMRI data will bridge mesoscopic scale neuronal activity at each cortical layer and large- scale cortico-cortical connectivity. Therefore, this study will help reveal the circuit-level mechanism of RSFC. Critically, to avoid influences of anesthesia, all multi-modal data will be measured in awake rats, which also enables direct translation of our results to human rsfMRI studies. Together, the proposed research will provide critically needed knowledge that can bridge neuronal and hemodynamic activities at rest across wide spatiotemporal scales. Such knowledge so far is impossible to gain from human studies, but will directly impact our understanding of human rsfMRI research. Finally, given the high clinical relevance of rsfMRI, the proposed research will be an essential step toward our long-term goal of establishing rsfMRI as a noninvasive tool for aiding diagnosis and/or evaluating treatment options for brain disorders.